Modular Extracorporeal Systems and Methods for Treating Blood-Borne Diseases

ABSTRACT

Extracorporeal systems and methods for treating blood-borne diseases in a subject or for developing drugs to treat blood-borne diseases include various environmental and treatment modules that can be tailored to a specific disease or infection. In certain embodiments of the systems and methods, a blood sample is treated with cold plasma and optionally with hydrostatic pressure, a pulsed electrical field, a pharmaceutical agent, microwave, centrifugation, sonification, radiation, or a combination thereof, under environmental conditions that are effective for the treatment.

PRIOR RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/737,930, filed Jan. 9, 2013, claims priority to U.S. ProvisionalApplication No. 61/584,453, filed Jan. 9, 2012, the contents of whichare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The treatment of blood-borne disease by pharmacological intervention islimited by pathogen resistance and drug toxicity; the drugs used toattack pathogens are often also toxic to healthy human cells andtissues. Pharmaceutical intervention is also limited by the constant andrapid evolution of pathogens, especially bacteria, to adapt and becomeresistant to drugs. Methicillin-resistant Staphylococcus aureus (“MRSA”)is especially pernicious and is gaining national and worldwide attentionas a priority for disease control efforts. A 2007 report in EmergingInfectious Diseases, a publication of the Centers for Disease Controland Prevention (CDC), estimated the number of MRSA infections inhospitals doubled nationwide, from approximately 127,000 in 1999 to278,000 in 2005, while at the same time annual deaths increased from11,000 to more than 17,000. Klein E, Smith D L, Laxminarayan R (2007),“Hospitalizations and Deaths Caused by Methicillin-ResistantStaphylococcus aureus, United States, 1999-2005,” Emerg. Infect. Dis.13(12):1840-6. However, most large pharmaceutical companies in theUnited States have abandoned basic research and development of newantibiotic drugs and are focusing their research and development effortselsewhere, despite the ever growing problem of antibiotic resistance.

Accordingly, there is a need to develop systems and methods toefficiently and effectively treat blood-borne diseases while minimizingtoxicity to the patient. There is also a need in the art for methods ofcombating resistant pathogens in a patient, either by remodelingcurrently existing pharmaceutical interventions and/or by developing newand effective drugs and/or physical agents. Also needed are systems andmethods to evaluate the efficacy of new antimicrobial drugs and physicalagents under specific conditions, as well as to evaluate the safe use ofantimicrobial drugs and treatment methods that were previously found tobe effective against microbial pathogens but that were deemed too toxicfor in vivo use.

SUMMARY OF THE DISCLOSURE

The invention meets one or more of these needs by providing systems andmethods to efficiently and effectively treat blood-borne diseases whileminimizing toxicity to the patient; methods of combating resistantpathogens in a patient by allowing for the use of currently existingdrugs at levels not possible through current in vivo treatment and bydeveloping new drugs or agents; and systems and methods to evaluate theefficacy of new antimicrobial drugs and physical agents under specificconditions, as well as to evaluate the safe use of antimicrobial drugsand treatment methods that were previously found to be effective againstmicrobial pathogens but that were deemed too toxic for in vivo use.

The invention may be implemented in a number of ways.

In one aspect, extracorporeal methods for treating a blood-borne diseaseinclude the steps of removing blood from a subject; contacting the bloodwith an anticoagulant; optionally separating the blood into two or moreblood fractions; modifying the environment of the blood or bloodfraction; treating the blood or blood fraction with cold plasma andhydrostatic pressure, a pulsed electrical field, a pharmaceutical agent,centrifugation, radiation, microwave, sonification, or a combinationthereof; and returning at least a portion of the blood or blood fractionto the subject. In some embodiments, the anticoagulant is heparin orsodium citrate. In certain embodiments, the blood is separated into ared blood cell fraction, a buffy coat fraction, a platelet fraction, aplasma fraction, or a combination thereof. In certain embodiments, someor all of the buffy coat portion is not returned to the subject. In someembodiments, the step of modifying the environment of the blood or ablood fraction comprises at least one of modifying the pH, modifying thetemperature, modifying the oxygenation, modifying the availablenutrients, modifying the carbon dioxide, modifying the osmolality, or acombination thereof.

In some embodiments, the treating step comprises treating the blood orblood fraction solely with cold plasma. In certain embodiments, the coldplasma treatment uses argon, helium, hydrogen, or nitrogen gases, or acombination thereof. The gases, in some embodiments, may be energizedwith the use of, for example, microwave, A/C or D/C electrical energy,radiowaves, laser or electron beams, or a combination thereof. Incertain embodiments, the cold plasma treatment may be implementedthrough the use of a non-thermal plasma torch, pencil, wand, pipe,honeycomb pipe array, mixer, or combination thereof. In certainembodiments, the treatment time with cold plasma is applied for about 10to about 200 minutes.

In some embodiments, the modifying the environment step compriseslowering the pH, and the treating step comprises treating the blood orblood fraction with at least two treatment modules. The at least twotreatment modules comprise treatment with cold plasma and treatment witha pharmaceutical agent that is acidic. The step of lowering the pH maycomprise, for example, contacting the blood or blood fraction withcarbonic acid, citric acid, hydrochloric acid, lactic acid, acetic acid,pyruvic acid, or a combination thereof. In other embodiments, themodifying the environment step comprises increasing the pH of the bloodor blood fraction, and the treating step comprises treating the blood orblood fraction with at least two treatment modules. The at least twotreatment modules comprise treatment with cold plasma and treatment witha pharmaceutical agent that is basic. The step of increasing the pH maycomprise, for example, contacting the blood with bicarbonate.

In certain embodiments, the temperature is modified to increase thereplication rate of the pathogen. In certain embodiments, the modifyingthe environment step comprises reducing the blood or blood fractiontemperature to about 30° C. to about 36° C. In other embodiments of themethods, the modifying the environment step comprises increasing theblood or blood fraction temperature to about 37° C. to about 42° C. Insome embodiments, the modifying the environment step comprisesoxygenating the blood or blood fraction. In other embodiments, themodifying the environment step comprises deoxygenating the blood orblood fraction.

The extracorporeal methods of the invention may include the step oftreating the blood or blood fraction with cold plasma and one or moreadditional steps such as a treating step that comprises treating theblood or blood fraction with a pharmaceutical agent that is effective inanaerobic conditions, such as, but not limited to, metronidazole,clindamycin, chloramphenicol, or combinations thereof.

In certain embodiments, the methods may include the additional steps ofmodifying the environment of the treated blood or blood fraction byoxygenating the treated blood or blood fraction; and then administeringto the blood or blood fraction a pharmaceutical agent effective inaerobic conditions, such as, but not limited to, daptomycin,azithromycin, silver, or combinations thereof.

In some embodiments, the step of modifying the environment comprisesadding glucose to the blood or blood fraction. In other embodiments, thestep of modifying the environment comprises reducing glucose in theblood or blood fraction.

In some embodiments, the step of modifying the environment comprisesincreasing the carbon dioxide in the blood or blood fraction. In certainembodiments, the treating step comprises treating the blood or bloodfraction with at least two treatment modules. The at least two treatmentmodules may comprise treatment with cold plasma and with hydrostaticpressure at a pressure range from about 50 MPa to about 1,000 MPa. Incertain embodiments, the step of modifying the environment alsocomprises reducing the temperature of the blood or blood fraction, andthe treating step comprises treating the blood or blood fraction withcold plasma and with hydrostatic pressure.

In some embodiments, the treating step comprises treating the blood orblood fraction with at least two treatment modules. The at least twotreatment modules may comprise treatment with cold plasma and treatingthe blood or blood fraction with a pulsed electrical field and apharmaceutical agent. In certain embodiments, the step of modifying theenvironment comprises reducing the blood or blood fraction temperature,and the treating step comprises treating the blood or blood fractionwith cold plasma and with a pulsed electrical field and a pharmaceuticalagent.

In some embodiments, the step of modifying the environment comprisesreducing the blood or blood fraction temperature, and the treating stepcomprises treating the blood or blood fraction with at least twotreatment modules. The at least two treatment modules may comprisetreatment with cold plasma and with microwaves.

In some embodiments, the treating step comprises treating the blood orblood fraction with cold plasma and by centrifugation and removal of thepathogens and/or infected cells.

In some embodiments, the treating step comprises treating the blood orblood fraction with at least two treatment modules. The at least twotreatment modules comprise treatment with cold plasma and by irradiatingthe blood or blood fraction by exposing the blood or blood fraction toX-ray, UV, IR, visible, laser, radiofrequency energy, or a combinationthereof. In certain embodiments, the step of irradiating the blood orblood fraction comprises exposing the blood or blood fraction to about50 to about 75 gray units.

In another aspect, the extracorporeal treatment methods of the inventionmay further comprise a step of removing toxins from the blood or bloodfraction before or after the treating step. The step of removing toxinsmay comprise, for example, filtering the blood or blood fraction,dialyzing the blood or blood fraction, chelating the blood or bloodfraction, absorbing toxins from the blood or blood fraction, or acombination thereof. In certain embodiments, the filtering stepcomprises directing the blood through a filter having an average poresize of about 0.3 to about 1.5 microns. In other embodiments, thefiltering step comprises directing the blood through an antibody capturemodule. In certain embodiments, an antibody capture module may comprisemonoclonal antibodies with magnetic nanoparticles.

In any of these aspects, the extracorporeal method can also include theadditional steps of: modifying the environment of the treated blood or ablood fraction; treating the blood or blood fraction with hydrostaticpressure, a pulsed electrical field, a pharmaceutical agent, microwave,centrifugation, radiation, sonification, cold plasma, or a combinationthereof; and optionally repeating any one of these steps.

In another aspect, methods for the extracorporeal treatment of MRSAinclude the steps of removing blood from a subject; contacting the bloodwith an anticoagulant; separating the blood into red blood cell, buffycoat, and plasma fractions; treating the blood or one or more bloodfractions with cold plasma; treating the red blood cell fraction with atleast one pharmaceutical module; treating the plasma fraction with aradiation module; subjecting both the red blood cell fraction and theplasma fraction to a filtration module; and returning at least a portionof the blood or blood fraction to the subject. In some embodiments, theat least one pharmaceutical module delivers silver ions. In someembodiments, the pharmaceutical module is combined with a pulsedelectrical field module to deliver gentamicin and daptomycin. In someembodiments, the pharmaceutical module is combined with a pulsedelectrical field module to deliver metronidazole and clindamycin underanaerobic conditions. In some embodiments, the methods include threepharmaceutical modules including one that delivers silver ions, one thatis combined with a pulsed electrical field module to deliver gentamicinand daptomycin, and one that is combined with a pulsed electrical fieldmodule to deliver metronidazole and clindamycin under anaerobicconditions

In another aspect, methods for the extracorporeal treatment of Bacillusanthracis include the steps of removing blood from a subject; contactingthe blood with an anticoagulant; separating the blood into red bloodcell, buffy coat, and plasma fractions; treating the blood or one ormore blood fractions with cold plasma; treating the red blood cellfraction with at least one pharmaceutical module; treating the plasmafraction with a high hydrostatic pressure module; subjecting both thered blood cell fraction and the plasma fraction to a filtration module;and returning at least a portion of the blood or blood fraction to thesubject. In some embodiments the pharmaceutical module delivers a highdose of cisplatin, mevastatin, or tetracyclines. In other embodiments,the pharmaceutical module is used in combination with a high hydrostaticcompression module to deliver a high dose of a quinolone antibiotic. Insome embodiments, the methods include four pharmaceutical modulesincluding one that delivers cisplatin, one that delivers mevastatin, onethat delivers tetracyclines, and one that is combined with an HHP moduleto deliver a quinolone antibiotic.

In another aspect, methods for the extracorporeal treatment of malariainclude the steps of removing blood from a subject; contacting the bloodwith an anticoagulant; separating the blood into red blood cell, buffycoat, and plasma fractions; treating the blood or one or more bloodfractions with cold plasma; treating the red blood cell fraction with atleast one pharmaceutical module; treating the plasma fraction with amicrowave module; subjecting both the red blood cell fraction and theplasma fraction to a filtration module; and returning at least a portionof the blood or blood fraction to the subject. In some embodiments thepharmaceutical module delivers a high dose of quinine, clindamycin,artemether, doxycycline, or a combination thereof. In other embodiments,the pharmaceutical module is used in combination with a low energymicrowave module. In some embodiments, the methods include threepharmaceutical modules including one that delivers quinine andclindamycin, one that delivers artemether, and one that deliversdoxycycline.

In another aspect, methods for the extracorporeal treatment ofhemorrhagic fever include the steps of removing blood from a subject;providing the subject with replacement platelets, white blood cells, redblood cells, and/or saline; contacting the removed blood with ananticoagulant; separating the blood into red blood cell, buffy coat, andplasma fractions; treating the blood or one or more blood fractions withcold plasma; treating the red blood cell fraction with at least onepharmaceutical module; treating the plasma fraction with a radiationmodule; subjecting both the red blood cell fraction and the plasmafraction to a filtration module; and returning at least a portion of theblood or blood fraction to the subject.

In another aspect of the invention, extracorporeal systems include ablood removal port; an anticoagulant module; a module adapted to modifythe environment of blood or a blood fraction; at least one treatmentmodule adapted to administer cold plasma, or at least two treatmentmodules to administer cold plasma and hydrostatic pressure, a pulsedelectrical field, a pharmaceutical agent, microwave, centrifugation, ora combination thereof; and a blood return port. In some embodiments, themodule for modifying the environment comprises an environmental module.In certain embodiments, the environmental module comprises at least oneof a pH modifying module, a deoxygenation module, and a temperaturecontrol module. In certain embodiments, the environmental module is apH-modifying module, and wherein one of the treatment modules is adaptedto administer a pharmaceutical agent that is effective at a certain pHrange. In certain other embodiments, the environmental moduledeoxygenates the blood or blood fraction, and wherein one of thetreatment modules is adapted to administer a pharmaceutical agent thatis effective in anaerobic conditions. In other embodiments, theenvironmental module decreases the temperature of the blood or bloodfraction, and wherein one of the treatment modules is adapted toadminister hydrostatic pressure, a pulsed electrical field, or acombination thereof.

In another aspect, systems for the extracorporeal treatment of MRSA mayinclude a blood removal port; an anticoagulant module; a separationmodule; a module adapted to modify the environment of blood or a bloodfraction; a treatment module adapted to administer cold plasma; at leastone treatment module adapted to administer a pharmaceutical agent,pulsed electric field, radiation, or a combination thereof; a filtrationmodule; and a blood return port.

In another aspect, systems for the extracorporeal treatment of Bacillusanthracis may include a blood removal port; an anticoagulant module; aseparation module; a module adapted to modify the environment of bloodor a blood fraction; a treatment module adapted to administer coldplasma; at least one treatment module adapted to administer hydrostaticpressure, a pharmaceutical agent, or a combination thereof a filtrationmodule; and a blood return port.

In another aspect, systems for the extracorporeal treatment of malariamay include a blood removal port; an anticoagulant module; a separationmodule; a module adapted to modify the environment of blood or a bloodfraction; a treatment module adapted to administer cold plasma; at leastone treatment module adapted to administer a pharmaceutical agent,microwave, or a combination thereof; a filtration module; and a bloodreturn port.

In another aspect, systems for the extracorporeal treatment ofhemorrhagic fever may include a blood removal port; an anticoagulantmodule; a separation module; a module adapted to modify the environmentof blood or a blood fraction; a treatment module adapted to administercold plasma; at least one treatment module adapted to administer apharmaceutical agent, radiation, or a combination thereof; a filtrationmodule; and a blood return port.

In another aspect of the invention, methods for developing a new drug ortreatment regimen in an extracorporeal system for the treatment of asubject include the steps of obtaining a blood sample that contains aknown concentration of pathogens; optionally separating the blood sampleinto a red blood cell fraction, a buffy coat fraction, a plasmafraction, or a combination thereof; modifying the environment of atleast a portion of the blood sample or a blood fraction; treating theblood sample or blood fraction with cold plasma; optionally treating theblood sample or blood fraction with hydrostatic pressure, a pulsedelectrical field, a pharmaceutical agent, microwave, centrifugation,radiation, sonification, or a combination thereof; and determining theconcentration of pathogens in the blood sample or blood fraction afterthe treating step or steps, wherein the treatment is successful for thepathogen if it eliminates or reduces the concentration of the pathogensin the blood sample or blood fraction when compared to the knownoriginal concentration in the sample. In certain embodiments, thesemethods further include the step of treating a subject with the drug ortreatment determined to be successful in the extracorporeal system.

In another aspect of the invention, extracorporeal systems fordeveloping a new drug or treatment regimen for the extracorporealtreatment of a subject include a first blood or blood fractioncollection chamber with a first inlet and a first outlet port; ananticoagulant module; a module to modify the environment of the blood orblood fraction; a treatment module adapted to administer cold plasma;optionally, at least one treatment module that is adapted to administerhydrostatic pressure, a pulsed electrical field, a pharmaceutical agent,microwave, centrifugation, radiation, sonification, cold plasma, or acombination thereof to the blood or blood fraction; and a second bloodor blood fraction collection chamber with a second inlet port and asecond outlet port. In certain embodiments, the extracorporeal systemsfurther include a module to separate the blood or blood fraction into ared blood cell fraction, a buffy coat fraction, a plasma fraction, or acombination thereof. In certain embodiments, the extracorporeal systemsfurther include a sensor or other device to determine the concentrationof pathogens in the blood sample or blood fraction in the first and/orthe second blood or blood fraction collection chamber.

In another aspect of the invention, methods for treatment of a bloodsample or blood fraction prior to transfusion to a subject include thesteps of obtaining a blood sample or blood fraction; optionallyseparating the blood sample or blood fraction into a red blood cellfraction, a buffy coat fraction, a plasma fraction, or a combinationthereof; modifying the environment of at least a portion of the bloodsample or the blood fraction; treating the blood sample or bloodfraction with cold plasma; optionally treating the blood sample or bloodfraction with hydrostatic pressure, a pulsed electrical field, apharmaceutical agent, microwave, centrifugation, radiation,sonification, or a combination thereof; and determining theconcentration of pathogens in the blood sample or blood fraction beforeand/or after the treating step.

In another aspect, extracorporeal systems for the treatment of a bloodsample or blood fraction prior to transfusion to a subject include afirst blood or blood fraction collection chamber with a first inlet anda first outlet port; an anticoagulant module; an optional module toseparate the blood or blood fraction into a red blood cell fraction, abuffy coat fraction, a plasma fraction, or a combination thereof; amodule to modify the environment of the blood or blood fraction; atreatment module adapted to administer cold plasma; optionally, at leastone treatment module that is adapted to administer hydrostatic pressure,a pulsed electrical field, a pharmaceutical agent, microwave,centrifugation, radiation, sonification, or a combination thereof to theblood or blood fraction; and a second blood or blood fraction collectionchamber with a second inlet port and a second outlet port. These systemsmay, in certain embodiments, also include a sensor to determine theconcentration of pathogens in the blood sample or blood fraction in thefirst and/or the second blood or blood fraction collection chamber.

In another aspect of the invention, devices for use in the systems ormethods disclosed herein are provided.

Additional features, advantages, and embodiments of the invention may beset forth or apparent from consideration of the following detaileddescription and claims. Moreover, it is to be understood that both theforegoing summary of the invention and the following detaileddescription are exemplary and intended to provide further explanationwithout limiting the scope of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a furtherunderstanding of the invention, are incorporated in and constitute apart of this specification, and illustrate embodiments of the inventionand together with the detailed description serve to explain theprinciples of the invention. No attempt is made to show structuraldetails of the invention in more detail than may be necessary for afundamental understanding of the invention and various ways in which itmay be practiced.

FIG. 1 is a schematic drawing illustrating an extracorporeal systemconstructed according to the principles of the invention including ananticoagulant module, a separation module, a temperature control module,a first environmental module (as a pretreatment module, expanded in thisdrawing to show several potential environmental modules), a cold plasmatreatment module, an optional additional treatment module (or modules),a toxin removal module, and a second environmental module (as aposttreatment module before the blood, blood fraction, or a portionthereof is returned to the patient). Both environmental modules mayinclude one or more modules for modifying the environment of the bloodor blood fraction, as discussed in detail below. In addition, althoughthe drawing includes the separation module prior to the firstenvironmental module, in other embodiments the separation module may notbe included in the system, may be included after the first environmentalmodule and before the treatment module or modules, or may be included asa treatment module.

FIG. 2 is a schematic drawing that illustrates an expanded view of anexemplary separation module of the invention. This embodimentillustrates the separation of the blood into two or more different bloodfractions, such as white blood cells, red blood cells, plasma, andpathogens. In certain embodiments, the pathogens are located with thebuffy coat component after centrifugation, and the buffy coat/pathogencomponent is eliminated from the fluid prior to returning the blood orblood fraction or a part thereof to the patient. In certain embodiments,the separation module is used to separate white cells from other bloodfractions, both for the purposes of treating hemorrhagic fevers and alsoto take advantage of new techniques available to enhance white cellfunction by extracorporeal treatments and then return the white bloodcells to the circulation in the patient.

FIG. 3 is a schematic drawing that illustrates an expanded view of anexemplary environmental module of the invention. This one embodimentincludes a temperature control module, an oxygen control module(including an oxygenating component and deoxygenating component), pHcontrol module (including pH-increasing component (bicarbonate) andpH-decreasing component (carbonic acid)), nutrient control module(including glucose-increasing component and glucose-decreasingcomponent), carbon dioxide control module, and osmolality controlmodule.

FIGS. 4 to 9 are schematic drawings that illustrate exemplary treatmentmodules of the invention that optionally may be combined with the coldplasma treatment module as shown in FIG. 1. FIG. 4 is a schematicdrawing showing an expanded view of an exemplary pharmaceutical moduleof the invention to administer an antimicrobial drug. Certainpharmaceutical agents require longer exposure times in order to beeffective against certain pathogens. Therefore, the coil shown in thisdrawing reflects an example of one embodiment in which a longer exposuretime to a pharmaceutical agent may be achieved. Similar extendedexposure times may be achieved by any methods known in the art such as,for example, directing the fluids to a reservoir or other container inorder to expose the pathogen for a particular period of time beforemoving to the next treatment or module.

FIG. 5 is a schematic drawing that illustrates an expanded view of anexemplary radiation module of the invention. This module may be disposedafter a separation module in which the blood is separated into a plasmacomponent and a blood cell component. The plasma component may besubjected to UV radiation, whereas the cellular component may besubjected to X-ray radiation.

FIG. 6 is a schematic drawing that illustrates an expanded view of oneembodiment of an exemplary high hydrostatic pressure (HHP) module of theinvention.

FIG. 7 is a schematic drawing that illustrates an expanded view of oneembodiment of an exemplary pulsed electrical field module of theinvention.

FIG. 8 is a schematic drawing that illustrates an expanded view of oneembodiment of an exemplary microwave module of the invention.

FIG. 9 is a schematic drawing that illustrates an expanded view of oneembodiment of an exemplary sonification module of the invention.

FIGS. 10 to 13 are schematic drawings illustrating several exemplarytoxin removal modules of the invention. FIG. 10 is a schematic drawingthat illustrates an expanded view of one embodiment of an exemplaryfiltration module of the invention. In this embodiment, the filtrationmodule comprises a disposable polymyxin cartridge

FIG. 11 is a schematic drawing that illustrates an expanded view of anexemplary dialysis module of the invention.

FIG. 12 is a schematic drawing that illustrates an expanded view of anexemplary chelation module of the invention.

FIG. 13 is a schematic drawing that illustrates an expanded view of anexemplary adsorption/absorption module of the invention.

FIG. 14 is a schematic drawing that illustrates an exemplary embodimentof an extracorporeal system of the invention particularly designed forthe treatment of MRSA. This embodiment includes an anticoagulant module,a separation module to separate the blood into a red blood cellfraction, a plasma fraction, and a buffy coat fraction comprising someof the pathogen (which is discarded and not returned to the patient).The red blood cell fraction is then subjected to a pretreatmentenvironmental module, a cold plasma module, and a pharmaceutical modulefor the delivery of silver ions to degrade bacterial capsule andmembrane (shown as component a). The red blood cell fraction is alsotreated with a combination of a pharmaceutical module and a pulsedelectric field (PEF) module under aerobic conditions (shown as componentb) for treatment with a high dose of gentamicin and daptomycin, as wellas a deoxygenation component using a low energy PEF to facilitateantibiotic penetration of the bacteria. The red blood cell fraction istreated with an oxygen removal module (shown as component c), and acombination of a pharmaceutical module designed for anaerobic conditionsand a PEF module for treatment with a high dose of metronidazole andclindamycin using a low energy PEF to facilitate antibiotic penetration(shown as component d). The plasma fraction, in addition to or insteadof the red blood cell fraction, may be subjected to treatment with coldplasma. The cold plasma treatment module(s) may be present before orafter any other treatment or environmental module rather than at thespecific location shown in this exemplary embodiment. The plasmafraction is subjected to UV radiation. Both the plasma fraction and thered blood cell fraction are filtered to remove pathogens and/orendotoxins, before proceeding to a post-treatment environmental moduleand returning to the patient.

FIG. 15 is a schematic drawing that illustrates an exemplary embodimentof an extracorporeal system of the invention particularly designed forthe treatment of Bacillus anthracis (anthrax). This embodiment includesan anticoagulant module, a separation module to separate the blood intoa red blood cell fraction, a plasma fraction, and a buffy coat fractioncomprising some of the pathogen (which is discarded and not returned tothe patient). The red blood cell fraction is then subjected to apretreatment environmental module, a cold plasma treatment module, and apharmaceutical module for the delivery of a high dose of cisplatin toinactivate lethal anthrax toxins (shown as component a). The red bloodcell fraction is also treated with another pharmaceutical module todeliver mevastatin to attenuate the anthrax infection progression (shownas component b), as well as another pharmaceutical module to delivertetracyclines, animal derived peptides, to kill both anthrax bacilli andspores (shown as component c). The red blood cell fraction is treatedwith a combination of a pharmaceutical module designed and a highhydrostatic pressure (HHP) module for treatment with a high dose ofquinolone antibiotic along with low level HHP for the synergistickilling of bacilli and spores (shown as component d). The plasmafraction, in addition to or instead of the red blood cell fraction, maybe subjected to treatment with cold plasma. The cold plasma treatmentmodule(s) may be present before or after any other treatment orenvironmental module rather than at the specific location shown in thisexemplary embodiment. The plasma fraction is subjected to an HHP modulewith high level HHP to sterilize the plasma of all bacilli and spores(shown as component e). Both the plasma fraction and the red blood cellfraction are filtered to remove pathogens and/or endotoxins, beforeproceeding to a post-treatment environmental module and returning to thepatient.

FIG. 16 is a schematic drawing that illustrates an exemplary embodimentof an extracorporeal system of the invention particularly designed forthe treatment of malaria. This embodiment includes an anticoagulantmodule, a separation module to separate the blood into a red blood cellfraction, a plasma fraction, and a buffy coat fraction comprising someof the pathogen (which is discarded and not returned to the patient).The red blood cell fraction is then subjected to a pretreatmentenvironmental module, a cold plasma treatment module, and apharmaceutical module for the delivery of a high dose of quinine andclindamycin (shown as component a). The red blood cell fraction is alsotreated with another pharmaceutical module to deliver a high dose ofartemether (shown as component b), as well as another pharmaceuticalmodule to deliver a high dose of doxycycline (shown as component c). Thered blood cell fraction is treated with a combination of apharmaceutical module and a microwave module for treatment with a lowenergy microwave exposure to further reduce parasite viability (shown ascomponent d). The plasma fraction, in addition to or instead of the redblood cell fraction, may be subjected to treatment with cold plasma. Thecold plasma treatment module(s) may be present before or after any othertreatment or environmental module rather than at the specific locationshown in this exemplary embodiment. The plasma fraction is subjected toa microwave module with high energy microwave exposure to sterilize theplasma of any parasites (shown as component e). Both the plasma fractionand the red blood cell fraction are filtered to remove pathogens and/orparasite debris, before proceeding to a post-treatment environmentalmodule and returning to the patient.

FIG. 17 is a schematic drawing that illustrates an exemplary embodimentof an extracorporeal system of the invention particularly designed forthe treatment of hemorrhagic fevers such as Dengue and Ebola. Thisembodiment includes an anticoagulant module, a separation module toseparate the blood into a red blood cell fraction, a plasma fraction,and a buffy coat fraction comprising some of the pathogen and infectedwhite blood cells (which is discarded and not returned to thepatient)(shown as component h). The red blood cell fraction is thensubjected to a pretreatment environmental module, a cold plasmatreatment module, and a pharmaceutical module for the delivery of anRNA-dependent polymerase inhibitor (shown as component a). The red bloodcell fraction is also treated with another pharmaceutical module todeliver human recombinant interferon to degrade the virus (shown ascomponent b), as well as another pharmaceutical module to deliver goatderived immunoglobulin specific to the hemorrhagic fever virus beingtreated (shown as step c). The red blood cell fraction is treated with apharmaceutical module to deliver activated C-protein to augment thesubject's natural anti-viral immune processes (shown as component d).The plasma fraction, in addition to or instead of the red blood cellfraction, may be subjected to treatment with cold plasma. The coldplasma treatment module(s) may be present before or after any othertreatment or environmental module rather than at the specific locationshown in this exemplary embodiment. The plasma fraction is subjected toa radiation module to deliver high energy UV radiation to sterilizeplasma of the virus (shown as component f). Both the plasma fraction andthe red blood cell fraction are subjected to a filtration module toremove specific viral endotoxins (shown as component e), beforeproceeding to a post-treatment environmental module and returning to thepatient. In addition, platelets, white blood cells, red blood cells,and/or saline may be administered to the patient as a replacement tocontrol hemorrhage and shock (shown as component g).

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the invention is not limited to the particularmethodology, protocols, and mechanical features, etc., described herein,as these may vary as the skilled artisan will recognize. It is also tobe understood that the terminology used herein is used for the purposeof describing particular embodiments only, and is not intended to limitthe scope of the invention. It also is be noted that as used herein andin the appended claims, the singular forms “a,” “an,” and “the” includethe plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a drug” is a reference to one or moredrugs and equivalents thereof known to those skilled in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which the invention pertains. The embodiments of theinvention and the various features and advantageous details thereof areexplained more fully with reference to the non-limiting embodiments andexamples that are described and/or illustrated in the accompanyingdrawings and detailed in the following description. It should be notedthat the features illustrated in the drawings are not necessarily drawnto scale, and features of one embodiment may be employed with otherembodiments as the skilled artisan would recognize, even if notexplicitly stated herein. Descriptions of well-known components andprocessing techniques may be omitted so as to not unnecessarily obscurethe embodiments of the invention. The examples used herein are intendedmerely to facilitate an understanding of ways in which the invention maybe practiced and to further enable those of skill in the art to practicethe embodiments of the invention. Accordingly, the examples andembodiments herein should not be construed as limiting the scope of theinvention, which is defined solely by the appended claims and applicablelaw.

As used herein, the terms “subject” and “patient” are usedinterchangeably to refer to any animal capable of blood removal andreturn. The subject or patient is preferably a mammal, and is mostpreferably a human, but also may include laboratory, pet, domestic, orlivestock animals.

Unless otherwise specified, the term “blood” refers to whole blood orany fraction(s) of whole blood including a red blood cell fraction, abuffy coat fraction, a platelet fraction, a plasma fraction, and anycombination of two or more of these fractions.

As used herein, the term “pathogen” includes any invading microorganismincluding, but not limited to, bacteria, viruses, protozoa, fungi,prions, prion-like particles, and other parasites. In certainembodiments, the pathogen includes, but is not limited to,Staphylococcus aureus, Escherichia coli, Streptococcus, Klebsiella,Enterobacter, Meningococcus, Treponema, and other bacteria. In certainother embodiments, the pathogen includes, but is not limited to, amalarial parasite, a trypanosomal parasite, and other parasites. In yetother certain embodiments, the pathogen includes, but is not limited to,human immunodeficiency virus, hepatitis viruses A, B, C, D, and E,herpesvirus, human papillomavirus, arbovirus, human T-lymphotropic virustype I, and West Nile virus and other viruses.

“Blood-borne disease” includes any disease in which a pathogen or a partthereof is located in the bloodstream. It can, but need not be a diseasecharacterized by infection of the blood itself (such as septicemia), aneoplastic disease (such as a leukemia), or an immune disorder.

A “drug,” “pharmaceutical agent,” or “antimicrobial agent,” as usedherein, includes any small molecule, gas, or biologic agent effective tokill or impair one or more pathogens. Notably, pharmaceutical agentsused herein do not require any minimum safety or toxicity thresholdbecause they are not administered directly to the subject, but ratherare administered extracorporeally. Accordingly, pharmaceutical agentsmay be used in the present methods at high concentrations for variableperiods of time as required by the agent and pathogen. In addition,certain combinations of pathogen and drug(s) may require very high dosesof the drug(s) to be delivered rapidly via pulsation techniques asopposed to steady state delivery of the drug to the pathogen over aperiod of time.

As used herein, the term “blood fraction” refers to a red blood cellfraction, a buffy coat fraction, a white blood cell fraction, a plateletfraction, a plasma fraction, or a combination thereof.

Embodiments of the invention provide modular extracorporeal methods andsystems, whereby blood is removed from a subject, the blood is subjectedto a customizable circuit of environmental, pharmaceutical, andnon-pharmaceutical interventions, and finally the blood is returned tothe subject. By moving the battlefield in the war against infection outof the body and into an extracorporeal system, many common patienttoxicity problems experienced with conventional in vivo antimicrobialtherapies will be reduced or eliminated. Because the extracorporealsystem can control environmental conditions in a way not possible invivo, the system will restore efficacy to drugs currently renderedineffective by resistance, and also permit the use of drugs that may beeffective against pathogens, but are intolerably toxic in vivo. Inaddition, the extracorporeal system can be used to treat blood (e.g.,from a blood bank) for use in transfusion to a different subject toensure that it is safe and free of pathogens. The extracorporeal systemcan also serve as a useful research tool for developing and screeningnew drugs and new drug classes and for optimizing the conditions underwhich the drugs are most effective.

Furthermore, the modular design of the device allows the treatment to beindividually tailored to a specific patient or a specific pathogen (See,e.g., FIGS. 14-17 for examples of some embodiments of the disclosedsystems for treatment of specific pathogens (although differentcombinations of modules and/or different order of the modules areenvisioned for each pathogen, in addition to the exemplary embodimentsshown in these figures)). This aspect will complement the currentlyevolving techniques of rapid DNA-based identification of microbes, suchas sepsis-producing organisms. It will now be possible to accuratelyidentify and specifically attack blood pathogens. The modules can beselected, customized, and sequenced to provide an optimized course for aspecific pathogen and/or to combat future mutations (e.g., resistance)of pathogens. In particular, the extracorporeal system can be used tocreate stressful environmental conditions to which the offendingmicrobes have little or no evolutionary experience. Thus, futuremicrobial resistance will be stymied. Without being bound by theory, theextracorporeal systems and methods described herein can be used, e.g.,as follows to combat particular mechanisms of microbial resistance topharmaceutical agents:

As a first mechanism, microorganisms develop the ability to activelypump or efflux antimicrobial drugs out of their systems. This pumpingrequires that the microbe expend energy to pump drugs out of theircells. The described methods, systems, and devices counteract this formof resistance by: a) administering a high concentration (e.g., higherthan the dose tolerated in vivo) of pharmaceutical agents that set up aconcentration gradient to force the diffusion of more drug into themicrobial cells; b) adjusting pH to towards the pKa of the antimicrobialdrug(s) to optimize both diffusion into, and ion-trapping within, thetarget cells; c) decreasing oxygen levels and/or decreasing glucoselevels to force the microbes to resort to less efficient pathways ofenergy production, which reduces energy availability for efflux pumpingand other resistance modes; and d) subjecting the blood or bloodfraction to low levels of pulsed electrical field discharge to enhancethe penetration of the pathogen by the antimicrobial agents and todefeat the active efflux pumping resistance mechanisms of the pathogen.

Second, microorganisms develop the ability to modify the structure ofcritical binding sites so that they have decreased affinity forpharmaceutical agents. This mechanism is similarly thwarted with theinventive principles because increasing the amount of drug inside themicrobial cell (as described in a)-d) above with respect to the firstmechanism) will partially compensate for decreased binding affinity ofany particular critical binding site.

Third, microorganisms produce a profusion of unmodified non-criticalbinding sites that serve to decoy and divert pharmaceutical agents awaycritical binding sites. This mechanism is similarly thwarted with theinventive principles because increasing the amount of drug inside themicrobial cell (as described in a)-d) above with respect to the firstmechanism) will partially compensate for the profusion of decoy bindingsites. Furthermore, the induction and production of large numbers ofdecoy binding sites is energy dependent, and the microbial cells willhave been forced into a state of reduced efficiency of energy productionby the modifying the controlled environment of the pathogen to containlow glucose levels and/or higher or lower oxygenation, for example.

Fourth, microorganisms produce enzymes that partially or completelyinactivate pharmaceutical agents. This mechanism is similarly thwartedbecause increasing the amount of drug inside the microbial cell (asdescribed in a)-d) above with respect to the first mechanism) will taxthe capacity of inactivating enzymes. Furthermore, microbial enzymeinduction and production are energy dependent, and the microbial cellshave been forced into a state of reduced energy production by creatingthe low glucose and/or modulated oxygen level environments.

Additionally, using drugs that block protein synthesis (e.g.,clindamycin) under anaerobic conditions will further degrade thepathogen's already distressed capacity to synthesize both antimicrobialdrug inactivating enzymes as well as the very enzymes that must beproduced to switch over to anaerobic and/or low glucose energyproduction.

Exemplary extracorporeal systems and methods of the invention fortreatment and for drug screening are described in further detail below.

I. Modular Extracorporeal Systems and Methods for Treating Blood-BorneDiseases

As shown in the schematically exemplary illustrated embodiments of thefigures, the invention provides modular extracorporeal systems andmethods for using the same to treat blood-borne diseases. Exemplaryblood-borne disease include, but are not limited to, bacterial diseases,conditions, or infections such as sepsis, Staphylococcus, Escherichiacoli, MRSA, Bacillus anthracis, syphilis, brucellosis, leptospirosis,tick-borne relapsing fever, Streptococcus, Klebsiella, Enterobacter,Meningococcus, and Treponema; viral diseases, conditions, or infectionssuch as HIV, hepatitis viruses A, B, C, D, and E, herpesvirus, humanpapillomavirus, arbovirus, human T-lymphotropic virus type I, viralhemorrhagic fever, and West Nile virus; protozoan diseases, conditions,or infections such as malaria, babesiosis, and diseases caused by atrypanosomal parasite; fungal diseases, conditions, or infections suchas coccidiomycosis and candidiasis; diseases, conditions, or infectionscaused by prions and prion-like particles such as Creutzfeldt-Jakobdisease (CJD); blood borne neoplastic diseases, conditions, orinfections such as the blast phase in leukemia; and diseases,conditions, or infections caused by other parasites such as worms andflukes.

A. Blood Removal

Blood can be removed from the subject by any conventional means, e.g.,catheter or port, known in the art including those known for blood andplasma donation and transfusion, dialysis treatment, vascular accessprocedures, and intravenous therapies. The system can include aperipheral line or a central line. As needed for stabilization of thesubject, volume replacement may be augmented by crystalloid solutions.Oxygenation capacity may be augmented (for the subject) as needed byblood transfusion or by semi-synthetic hemoglobin preparations.

B. Anti-Coagulant

The disclosed systems may involve providing an anti-coagulant to theblood after removal to ensure that the blood can circulate through thecircuit essentially unimpeded by clotting. Any anticoagulant known inthe art may be used including those known for dialysis and/orhemofiltration. In some embodiments, the anticoagulant is heparin orsodium citrate. When the anticoagulant is sodium citrate, addition ofthe anticoagulant module may also serve as the environmental module, assodium citrate will modify the environment of the blood by modifying thepH.

C. Cell Separation and Pathogen Removal Module

The cell separation and pathogen removal module (also referred to hereinas separation module) separates whole blood into two or more fractions.The separation module may be used in the present methods before or afteran environmental module, treatment module, or toxin removal module,depending on the goal of the separation (e.g., separation of bloodfractions to enable the most effective treatment without harm, orseparating out particular pathogens from specific blood fractions). Theblood fractions can be, for example, a red blood cell fraction, a buffycoat fraction, a platelet fraction, a plasma fraction, pathogenfraction(s), and any combination of two or more of these fractions.Separation can be accomplished by any method known in the art, such asthose methods used by blood banks (centrifugal or filtration based). Inanother embodiment, separation utilizes tangential flow and/orimmunological separation methods.

In certain embodiments, the separation module may be used as a treatmentmodule. In one embodiment, separation is accomplished by low speedcentrifugation, high speed centrifugation, or ultracentrifugation. Incertain embodiments, the low speed centrifugation, high speedcentrifugation, or ultracentrifugation is used to sequester pathogensaccording to their size and weight for removal from the extracorporealcircuit. Any centrifugation speed known in the art for separation ofcell types may be used. In some embodiments, centrifugation at about11,000 rpm to about 14,000 rpm, about 11,000 rpm to about 13,000 rpm, orabout 12,000 rpm to about 14,000 rpm may be used to separate uninfectedblood cells from infected blood cells. In certain embodiments, thesamples are centrifuged at about 12,000 rpm to about 13,000 rpm. Inother embodiments of the systems, high speed centrifugation at about20,000 rpm to about 40,000 rpm, about 20,000 rpm to about 30,000 rpm, orabout 30,000 to about 40,000 may be used to separate bacteria from bloodcells. In certain embodiments, the samples are centrifuged at about30,000 rpm. In other embodiments of the systems, ultra-centrifugation atabout 60,000 rpm to about 80,000 rpm, about 60,000 rpm to about 70,000rpm, or about 70,000 rpm to about 80,000 rpm may be used to separateviruses from blood cells. In certain embodiments, centrifugation atabout 70,000 rpm is used. Higher speeds may be used to separate outmolecules that are smaller in size than viruses.

In one embodiment, a buffy coat fraction is separated from the wholeblood. The buffy coat primarily consists of white cells and bacteriathat spin out into a layer between the red blood cell fraction and theplasma fraction. The buffy coat fraction may be circulated through theextracorporeal system (alone or in combination with other bloodfractions) and returned to the patient.

In another embodiment, the buffy coat fraction is separated from thewhole blood and not returned to the patient. Without being bound bytheory, the buffy coat fraction is believed to contain pathogens thathave been engulfed or partially engulfed by white blood cells byphagocytosis. The engulfed pathogens pose a serious threat in vivobecause they are largely protected from pharmaceuticals, e.g.,antibiotics, until the engulfing white cell disintegrates therebyreleasing the still viable and potentially drug-resistant pathogens backinto the bloodstream. In septicemia, the majority of blood-bornebacteria will often be trapped in the buffy coat fraction. Accordingly,in one embodiment, the buffy coat fraction is isolated and discarded. Bydiscarding the buffy coat fraction, the pathogen load of the bloodreturning to the patient can be significantly decreased. Moreover,destroying bacteria creates harmful endotoxins which themselves elicitan immune response. By removing the bacteria essentially intact, asopposed to destroying them within the extracorporeal circuit, endotoxinproduction and cytokine release is greatly reduced or eliminated, as isthe need for larger amounts of toxic drugs. Although a large portion ofthe pathogen load may be eliminated by discarding the buffy coatfraction, the pathogen load may be further diminished by subjecting theremaining blood fractions to one or more treatment modules, incombination with one or more environmental modules.

Although there is a concomitant loss of white cells associated withbuffy coat disposal, approximately 20% of the white cells will remain inthe red blood cell fraction, and even more are in reserve within thepatient's reticuloendothelial system and marginated along the vasculartree.

In another embodiment, the buffy coat fraction, or a sample thereof canbe used for diagnostics using conventional equipment. For example, asample of the buffy coat can be examined microscopically to identify thepathogen(s). With increasing advances in DNA-typing, it will be possibleto use a buffy coat sample to definitively identify not only the generictype of pathogen, but the precise genotype of the strain and resistancepattern. This typing can guide the specific treatment needed for thefurther processing of the blood or blood fractions in the extracorporealsystem disclosed herein to most effectively treat the particularpathogen(s) present.

D. Environmental Modules

One or more environmental modules (also referred to as “environmentalcontrol modules”) can be included in the extracorporeal system. As usedherein, the phrase “modifying the environment” of blood or a bloodfraction refers to changing the conditions of the blood or bloodfraction from the conditions under which the blood exists in the body,including, but not limited to, changing one or more external factors toaffect the sample. For example, in certain embodiments, modifying theenvironment of blood or a blood fraction may include modifying the pH,modifying the temperature, modifying the oxygenation, modifying theavailable nutrients, modifying the carbon dioxide, modifying theosmolality, or a combination thereof.

The environmental module(s) can be included before the treatment module(as a “pre-treatment,” as shown, e.g., in FIG. 1) or simultaneously withthe treatment module, in order to provide the appropriate environmentfor a treatment module to be safe and effective. The environmentalmodule(s) also may be interspersed between any two treatment modules, toensure that the environment for each of the treatment modules isoptimized for safety and efficacy. In addition, an environmental modulemay be included after any treatment modules to adjust the blood or bloodfraction toward physiological conditions before returning the blood orblood fraction to the subject (as a “post-treatment,” as shown, e.g., inFIG. 1).

The environmental control modules can be included once, more than once,or not at all in the system, depending on the particular treatmentmethod to be used. The invention also provides for sequential oppositemodules, that is, a module that increases a particular property followedby one that decreases that property or vice versa. For example, FIG. 3shows a module of high oxygen followed by a module of low oxygen or viceversa, a module of high glucose followed by a module of low glucose orvice versa, a module of high pH followed by a module of low pH or viceversa, and/or a module of high temperature followed by a module of lowtemperature or vice versa. These opposite modules can be coordinated toprovide a module effecting aerobic conditions and a module effectinganaerobic conditions (in either order). Complementary pharmaceuticalmodules can be paired with these environmental stages. When the systemincludes pairing a pharmaceutical agent to particular environmentalconditions, the environmental adjustment and the pharmaceuticaladministration can be performed in either order or essentiallysimultaneously.

Any or all of the environmental control modules may also include a meansfor monitoring the environmental conditions using equipment known in theart. Providing real-time monitoring of the environmental conditionsallows for adjustment of the conditions during circuit operation asnecessary to optimize safety and efficacy. Aspects of the sequentialopposite module concept are discussed in more detail below.

1. Temperature Control Module

Temperature elevation has been previously attempted both in vivo and invitro circuits to directly kill pathogens by increasing the temperature(e.g., above 40° C.). This temperature modulation has not proved to beuseful for in vivo treatment, as temperatures high enough to killpathogenic organisms also kill human blood cells. In directcontradiction to previous attempts to kill pathogens, one embodiment ofthe temperature module in the disclosed extracorporeal system ismodulated to increase the replication rate of the pathogens, which inturn renders them more susceptible to other means of attack (e.g.,pharmaceutical, radiation, high hydrostatic pressure compression, andpulsed electrical field treatment modules).

Optimal blood culture growth rates for human blood pathogens varyanywhere from about 20° C. to about 40° C. Many pathogens thrive attemperatures of about 35° C. to about 40° C. Thus, in some embodiments,the temperature is increased to about 35° C. to about 40° C., about 37°C. to about 42° C., about 37° C. to about 40° C., about 37° C. to about39° C., or about 38° C.

In other embodiments, the temperature is decreased to about 20° C. toabout 35° C., about 20° C. to about 30° C., about 25° C. to about 35°C., or about 30° C. to about 36° C. MRSA, for example, often lives andrapidly grows on human skin that may be ten degrees or more lower than37° C. Some Listeria bacteria actually grow best when incubated at closeto 20° C.

In addition, temperature control modules may be used prior to orsimultaneously with a treatment module, to provide the synchronizedcontrolled cooling of the extracorporeal blood or blood fraction, whichwill be necessary in certain treatment modules. For example, the highhydrostatic pressure module, the pulsed electrical field treatmentmodule, the sonification module, and the radiation module may cause somedegree of fluid warming that is dependent upon the intensity andduration of the treatment utilized. Therefore, temperature module(s) maybe used, for example, to lower the temperature of the blood or bloodfraction about 5° C. to about 25° C., about 5° C. to about 15° C., about10° C. to about 25° C., about 5° C. to about 10° C., about 8° C. toabout 12° C., or about 8° C. to about 10° C.

2. Oxygen Control Module

Human blood cells are remarkably tolerant of dramatic changes inoxygen-concentration, and so the tolerable oxygen concentrations will beessentially unlimited. Nevertheless, preferred oxygen concentrationranges are provided for guidance. For anaerobic conditions, the oxygenconcentration can be less than about 50, less than about 40, less thanabout 30, less than about 20, less than about 10, less than about 5, orless than about 1 mm Hg pressure. In one embodiment, the oxygenconcentration is about 0.5 to about 40 mm Hg pressure. For aerobicconditions, the oxygen concentration can be about 50 to about 150, about100 to about 150, or about 100 mm Hg pressure.

a) Increasing Oxygen

As discussed above, the environmental module of the disclosed system mayinclude an oxygenation component. The oxygenation component can involveany method or device known in the art to increase the oxygen content ofthe blood. Exemplary oxygenation components include, but are not limitedto, a micro-bubbler device, membrane diffusion devices, and devices asdescribed in, e.g., U.S. Pat. No. 5,104,373.

Without being bound by a particular theory, it is believed that oxygenenrichment is advantageous for several reasons. First, increasingcircuit oxygen levels will increase the replication rate for aerobicbacteria. This is significant because rapidly dividing cells are mostsusceptible to pharmaceuticals and radiation. Second, oxygen enrichmentforces facultative anaerobic and anaerobic bacteria to utilize aerobicpathways of energy metabolism, thus allowing drugs that block aerobicmetabolism to be more effective in attacking the bacteria's mostefficient energy producing systems. This is important as multi-drugresistant pathogens (e.g., MRSA, E. coli), many other pathogenicbacteria, and many non-bacterial disease-causing microbes arefacultative and can adapt to environments that are oxygen rich andoxygen poor. These facultative organisms are among the most threatening,as they are rapidly developing resistance to nearly all currentantibiotic classes when used in traditional in vivo methods at theaccepted concentrations. Additionally, raising oxygen levels decreasesthe resistance of MRSA to vancomycin, as well as restoring theeffectiveness of aminoglycosides against resistant E. coli. In addition,certain drugs, including but not limited to carboxyquinolones (e.g.,ciprofloxacin, levofloxacin, moxifloxacin), aminoglycosides,trimethoprim, and nitrofurantoin, have increased antimicrobial activityin high oxygen environments. Lastly, oxygen enrichment is useful toreplace circuit oxygen that will naturally decrease due to metabolism byblood cells within the circuit.

b) Decreasing Oxygen

Oxygen may be removed by any method or device known in the art. In oneembodiment, the “deoxygenating component” can be inherent to the systembecause oxygen concentration in the circuit will naturally decrease dueto metabolism by blood cells. As needed, further reduction in oxygen canbe accomplished by, e.g., a commercially available membrane diffusiondevice similar to those used to decrease blood oxygen for long termstorage in blood banking operations.

Without being bound by theory, it is believed that oxygen deprivation isadvantageous for several reasons. First, decreasing circuit oxygenlevels will increase the replication rate for predominantly anaerobicpathogens. As explained above, this is significant because rapidlydividing cells are most susceptible to treatment and destruction bypharmaceuticals and radiation. Most pathogenic anaerobic bacteria willgrow at levels of oxygen between about 1% to about 8%. Second, oxygendeprivation forces facultative anaerobic bacteria to utilize theirdefault survival energy producing fermentation pathways that allow themto survive under conditions of severe environmental stress. This willallow attack with alternative antimicrobial drugs that are effectiveagainst the bacteria's anaerobic stress survival pathways such asmetronidazole which uncouples anaerobic oxidative decarboxylation byacting as an electron sink and is rapidly lethal to anaerobes andfacultative anaerobes alike. Furthermore, metronidazole has activemetabolites that attack and destroy bacterial DNA. Industrial fermentingand water treatment operations have discovered natural plant andbacterial fermentation inhibiting chemicals, which may also be useful inthis system.

Sequential circuit exposures to alternating oxygen levels will allow forthe pathogens to be controlled in a manner that both optimally increasestheir reproduction rate, as well as forcing them to utilize specificmetabolic survival pathways that will then allow attack via specificantimicrobials that are most effective against anaerobic or aerobicmetabolic pathways (See e.g., FIG. 14). The sequential circuit exposurescan force the pathogen into less efficient metabolic pathways, permitattack of primary and secondary metabolic pathways independently, andovercome and/or evade the pathogen's evolutionary adaptive capabilities.Thus, the control methodology provides both new and effective techniquesfor combating bacterial hemosepsis and other blood pathogens.

3. pH Control Module

Controlling the pH of the blood provides the unique opportunity tomodulate the pH to coordinate with the pKa of the pharmaceutical agentto be administered. Without being bound by theory, it is believed thatmodulating pH according to the drug's pKa (i.e., coordinating the pHmodule and the treatment module) helps the drug to penetrate thepathogen membranes (e.g., bacterial cell walls) and enter the cytoplasm.Once inside, the drugs are re-ionized and trapped inside the pathogen,where they will continue to drive the concentration gradient to promotefurther antimicrobial diffusion into the pathogen. For acidic drugs, thepH of the blood or blood fraction should be decreased. Examples ofacidic drugs include, but are not limited to, finafloxacin,delafloxacin, beta-lactams (e.g., oxacillin), fusidic acid, andrifampicin. For basic drugs, the pH of the blood or blood fractionshould be increased. One non-limiting example of a basic drug isvancomycin which is capable of killing S. aureus under basic conditionsbetter than under slightly acidic conditions. Complete deionization isnot required; simply increasing the deionization will increasepermeability, and thus efficacy. Table 1 provides a list of exemplarydrugs and their respective pKa values, which would inform theappropriate pH conditions. This list is not intended to be limiting, andthe pKa value for any drug is readily available (see, e.g., the UnitedStates Pharmacopeia (USP)) and/or ascertainable.

TABLE 1 Drug pKa Amikacin 12.7 Amoxicillin 2.4 Amphotericin 2.5Azithromycin 8.1 Aztreonam 0.5 2.6 3.7 Ceftazidime 1.8 2.7 4.1Ceftriaxone sodium (COOH) 3 (NH₂) 3.2 Cefotaxime sodium 3.8 Cefoxitinsodium 2.2 Cefuroxime sodium 2.5 Cephalexin 3.2 Ciprofloxalin 6.1 8.6Clindamycin 7.5 Colistin 12.1 Colistimethate 3.9 Daptomycin 5.3 Asp-34.2 Asp-7 1.5 Asp-9 3.8 mGlu-12 4.6 Kyn-13 1.3 Gentamycin 8.2 13.2Imipenem 3.2 9.9 Levofloxacin (carboxylic group) 5.5 (piperazinyl group)8 6.8 Linezolid 5.0 1.7 Lincomycin 7.5 Metronidazole 2.5 Methicillin 3.0Minecycline 7.8 Naladixic acid 6 Norfloxacin 6.3 8.8 Penicillin G 2.8Polymixin 8.9 Polymixin B sulfate 12.0 Quinine 8.1 Rifampicin (-hydroxy)1.7 (3-piperazine nitrogen) 7.9 Sulfoxazole 5.0 Ticarcillin 3.0Tigecycline 4.4 Tobramycin 6.7 8.3 9.9 Trimethoprim 6.6 Trovafloxacin(COOH) 5.9 (NH₂) 8.1

The opportunity to modulate pH is uniquely possible in an extracorporealsystem or method of the invention, where the pH of the blood or bloodfraction can be modified to, e.g., pH values between about 4.5 to about9. In contrast, changing the pH in vivo is not feasible because humanblood pH is tightly regulated at a pH of about 7.4, and significantdeviations from this value are not tolerated. Similarly, although someprior art has suggested imposing dramatic changes to blood pH ex vivo tokill pathogens directly, such methods have proved ineffectual and/ordetrimental to blood cells, other critical tissues, and essential humanenzyme functions.

The pH of the blood or blood fraction can be decreased by anyphysiologically acceptable acidic compound including, but not limitedto, carbonic acid, ammonium chloride, citric acid, hydrochloric acid,lactic acid, acetic acid, and pyruvic acid. Conversely, the pH can beincreased by any physiologically acceptable basic compound such asbicarbonate or a stronger base. Just as high concentrations ofpharmaceutical agents will be tolerated extracorporeally as explained infurther detail below, high concentrations of pH adjusters will besimilarly tolerated ex vivo.

In some embodiments, modifying the pH can restore efficacy to previouslyapproved, but now ineffectual drugs in the antibiotic arsenal. Forexample, aminoglycoside antibiotics now fail to kill resistant strainsof E. coli under low pH conditions. Both the influx of the antibioticinto the bacterial cytoplasm and the ribosomal binding of theaminoglycoside are impaired at low pH conditions. The extracorporealsystem can couple increased pH with aminoglycoside antibiotics (andmoreover, the antibiotics can be administered at concentrations higherthan those acceptable in vivo as explained below). Further discussion ofdrugs that may be useful in the disclosed extracorporeal system isprovided below.

Also, modulating pH (particularly decreasing pH) can also serve asanother form of environmental stress that can be inflicted on thepathogenic organism. A pathogen can be subjected to a combination ofconditions rarely, if ever, seen in vivo, conditions under which thepathogen is evolutionarily ill-equipped. For example, a commonhemosepsis environment in vivo is often characterized by low pH, highpartial pressure of oxygen, and high glucose. By changing one or more ofthese variables in the disclosed extracorporeal system, the pathogen isexposed to unnatural, unfamiliar conditions that facilitate thereduction and/or elimination of the pathogen.

4. Nutrient Control Module

Pathogens are sensitive to the levels of carbohydrates (e.g., glucose),amino acids (e.g., tryptophan), iron, other trace elements, and otherkey nutrients in their environments. By default, many pathogens useglucose as a primary substrate for metabolism. In particular, duringsepsis, blood glucose is often higher than normal and so is readilyavailable. By manipulating the nutrient environment of the blood orblood fraction, one can direct the pathogen toward non-glucose metabolicpathways, which are often also anaerobic pathways (i.e., fermentationpathways). Additionally, it will be desirable to substitute othernon-glucose sugars such as lactose, pentose sugars, mannitol, and otherenergy substrates in order to expose alternative microbial metabolicpathways to attack. Forcing bacteria to use their alternate survivalmetabolic pathways invites the use of different antimicrobial agents(e.g., fermentation-blocking compounds) that attack these alternativemetabolic pathways. In some embodiments, the glucose levels will beabout 0.01 to about 300, 0.01 to about 5, about 5 to about 10, about 10to about 20, about 20 to about 40, about 40 to about 100, or about 100to about 300 mg/dL.

a) Increasing Nutrients

Increasing nutrients, e.g., glucose and iron concentrations can increasethe replication rate, thus increasing susceptibility to pharmaceuticalagents and radiation as discussed above. Paradoxically, somemicroorganisms will switch to fermentation-based energy production whenplaced in a high glucose environment even in the presence of oxygen.

b) Decreasing Nutrients

Significantly lowering glucose in vivo is lethal to human beings, butnot to blood cells. Thus, decreasing glucose is an environmentalcondition exclusively available in an extracorporeal system. Whenstressed by a low glucose environment, resistant pathogens must activatealternative non-glucose utilizing pathways to make energy, albeit lessefficiently. This involves synthesizing new enzymes to drive the lowglucose survival energy pathways.

The extracorporeal systems and methods of the invention may includedirecting the pathogens toward such alternative metabolic pathways, andwhile under such alternative metabolic conditions, administering proteinsynthesis inhibiting drugs to target the pathogens in this vulnerablestate. Multiple potential anti-metabolic drugs exist that can interferewith many aspects of microbial metabolism, but they are not effectiveagainst resistant pathogens under normal or elevated blood glucoseconditions. Thus, the extracorporeal systems and methods of theinvention provide a unique opportunity to use current anti-metabolicdrugs such as sulfoxazole, trimethoprim, various chemotherapeuticmedicines, anti-fermentation agents, protein synthesis inhibiting drugs,as well as drugs not otherwise available for treatments.

5. Carbon Dioxide Control Module

The carbon dioxide module may be included in the disclosedextracorporeal system as an environmental module and/or as a treatmentmodule. Carbon dioxide (CO₂) is a non-toxic gas with antimicrobialproperties. Desirably, CO₂ can be easily added and removed bymanipulating the pressure and temperature of a solution. Theantimicrobial effectiveness of CO₂ depends on the pressure andtemperature, but is also affected by the state of CO₂ (i.e., gas, liquidor supercritical fluid (SCF)) and the concentration of dissolved CO₂.The SCF region for CO₂ is above the critical temperature of 31° C. (Tc)and critical pressure of 7.4 MPa (Pc). SC CO₂ has properties betweenliquid and gas with higher dissolving and penetrating powers thatfacilitate entry through cell walls and into spores where CO₂ couldlower the pH or even extract the contents of microbial cell. Theantimicrobial action is primarily due to oxidation of the outer cellmembranes of vegetative bacteria, endospores, yeast, and molds.

CO₂ may be added to the blood or blood fraction by any methods known toone of ordinary skill in the art. For example, in some embodiments, theCO₂ is added to solution by: a) bubbling CO₂ in a vessel at lowtemperature and at slightly elevated pressures; b) adding CO₂ to theheadspace of a compressible container and then exposing to highpressure; and c) using CO₂ as the pressuring medium by injecting into avessel at high pressure. A variation of this technique is bubbling CO₂through a microfilter producing microbubbles that stay suspended in thesolution.

In certain embodiments, the carbon dioxide module is used as anenvironmental module in conjunction with a treatment module. Forexample, microbial inactivation by high pressure (HHP), discussed below,increases with adding CO₂, increasing temperature, pressure, and/ortime. Accordingly, in certain embodiments, the carbon dioxide module isused sequentially or simultaneously with an HHP module to maximize theantimicrobial effect.

In other embodiments, the carbon dioxide module is used in thisextracorporeal system as a treatment module. CO₂ can be easily added andremoved by manipulating the pressure and temperature of a solution. Thesolubility of CO₂ in water is expressed in volumes of dissolved CO₂ perunit volume of water, and the solubility increases with pressure anddecreases with temperature. In one embodiment, CO₂ enhanced theinactivation of Listeria when added at a carbonation volume of about 2at about 35° C., and pressure of up to about 350 MPa for about 300seconds.

6. Osmolality Module

In certain embodiments, the disclosed modular extracorporeal system mayinclude a module that affects the osmolality of the fluid. Theosmolality of any given blood sample may be different, depending on theparticular health condition of the subject. In certain embodiments, theosmolality may be modified with the use of a hypertonic solution anddistilled water.

In certain embodiments, the osmolality module is used as anenvironmental module in conjunction with a treatment module. Forexample, in certain embodiments, the osmolality module is usedsequentially or simultaneously with an HHP module or a PEF module inorder to maximize the antimicrobial effect. Similarly, the osmolalitymodule may be used after any treatment in order to restore the fluid toa condition suitable for transfer back to the patient.

E. Treatment Module

The disclosed modular extracorporeal system can include one or moretreatment modules which may include a pharmaceutical module, a radiationmodule, a hydrostatic compression module, a cell/pathogen separationmodule as described above, a pulsed electrical field module, a microwavemodule, and/or a sonification module (see, e.g., FIGS. 4-9).

1. Cold Plasma Module

In certain embodiments, the extracorporeal system of the invention mayinclude one or more cold plasma (non-thermal plasma) modules (see, e.g.,FIG. 1). Plasma is an ionized gas also referred to as the fourth stateof matter, and it consists of free-moving superheated electrons andions. It is a major component of starts such as the sun. Plasma isformed when gas is heated sufficiently for atoms to collide with eachother and displace their electrons. Cold plasma or non-thermal plasmasconsists of electrons that are superheated to thousands of degrees, butthey are produced by ionizing far less molecules than the high energyplasmas found on the stars. This heating produces a plasma in which theheat of the plasma is distributed to the many non-ionized molecules,which in effect functions as a heat sink resulting in a cool or at mostlukewarm plasma (about 95° F. to about 104° F.).

In the field of plasma, the temperature of the electrons and the bulkgas and/or the surrounding pressure is considered. In anatmospheric-pressure plasma device, extreme conditions are not required,but low temperatures occur. Atmospheric-pressure plasma is generated bycorona discharge, dielectric barrier discharge, or plasma jet. Mostcommonly used are radio frequency (rf) or microwave (mw) excited plasmasources. Radio-frequency (rf)-driven plasma jets can be used for studieson treatment of food related materials. Such a plasma source consists ofa needle electrode in the center of a ceramic nozzle and a groundedouter electrode. The rf voltage (e.g., f=27.12 MHz, P=20 W) is coupledvia a matching network to the needle electrode. The gas flows betweenthe electrodes, is ionized, and then ejected from the source. Thegenerated plasma contains chemical species, charged species, radicals,heat, and UV in different concentrations. The concentrations of thereagents are dependent on the process parameters and the gas used.

The presence of UV emitting species, charged particles, and freeradicals provides an antimicrobial effect to the plasma. Cold ornon-thermal plasmas kill bacteria by attacking the microbial surfacestructures as well as the microbial nucleic acids without harming humantissues. Cold plasma torches have been successfully employed to killdrug resistant bacteria in petri dish culture and in biofilms. Inparticular, the capability of non-thermal atmospheric plasmas toinactivate vegetative cells, including gram-negative and gram-positivebacteria, yeast, fungi, biofilm formers, and endospores has beendemonstrated. Treatment with non-thermal plasma is particularly suitedto the treatment of gram-negative bacteria, such as Escherichia coli andPseudomonas species, which are extremely sensitive to cold plasmatreatments. A non-limiting example of a gram-positive bacteria that issensitive to cold plasma treatment is Staphylococcus aureus.

In certain embodiments, the cold plasma treatment module of the modularextracorporeal system utilizes argon, helium, hydrogen, or nitrogengases, or a combination thereof. The gases may be energized in someembodiments with the use of, for example, microwave, A/C or D/Celectrical energy, radiowaves, laser or electron beams, or a combinationthereof. The cold plasma treatment may be implemented in the disclosedextracorporeal system, for example, through the use of a non-thermalplasma torch, pencil, wand, pipe, honeycomb pipe array, mixer, orcombination thereof. In certain embodiments, the treatment time withcold plasma is applied for about 45 seconds to about 400 minutes, about1 to about 400 minutes, about 5 to about 400 minutes, about 5 to about300 minutes, about 10 to about 300 minutes, about 5 to about 200minutes, or about 10 to about 200 minutes.

As discussed above with respect to certain exemplary embodiments of theextracorporeal systems, a blood sample or any fraction thereof (e.g.,plasma fraction, red blood cell fraction) may be subjected to treatmentwith cold plasma. The cold plasma treatment module(s) may be located inthe system sequentially or simultaneously coupled with any othertreatment module where appropriate, and/or sequentially orsimultaneously coupled with any environmental module.

The cold plasma treatment module may be used in the extracorporealsystems of the present invention for therapeutic treatment for bothhuman and veterinary uses. Cold plasma treatment in the presentextracorporeal systems also may be used as a research tool to assess andoptimize the conditions for treatment of microbial biofilmsintentionally cultured in the extracorporeal circuit.

2. Pharmaceutical Module

The modular extracorporeal system can include one or more pharmaceuticalmodules (see, e.g., FIG. 4). Each pharmaceutical module may be pairedwith the particular environmental conditions at that point in theextracorporeal system of the invention. Because in vivo toxicity is notapplicable to the extracorporeal system, drugs may be administered athigh concentrations, even concentration higher than those tolerated invivo. For example, linezolid is a promising, relatively new antibioticeffective against resistant bacteria, but toxic side effects havelimited its use. But in an extracorporeal system, linezolid and otherdrugs may be used without causing toxic side effect. Also, the system ofthe invention allows for the use of agents that have no tolerable invivo dose. In other words, drugs that are effective to kill pathogensbut are simply too lethal to use in vivo may now be employedextracorporeally in the disclosed systems. Such toxic drugs includehistorically toxic substances such as arsenic as well as drug candidatesthat have failed or will fail in clinical trials. Table 2 provides anon-limiting list of certain examples of drugs that have toxic effectsor limitations when used in vivo, but that would be suitable for use inthe disclosed extracorporeal system.

TABLE 2 Spectrum, Issues with Respect to In vivo Drug activity Toxicityor Side Effects Amikacin Gram negative, Nephrotoxicity, ototoxicitybactericidal Gentamicin Gram negative, Nephrotoxicity, ototoxicitybactericidal Tobramicin Gram negative, Nephrotoxicity, ototoxicitybactericidal Trovafloxacin Broad, Histamine release, Idiosyncraticbactericidal reactions Moxifloxacin Broad, Increase in dose limited dueto bactericidal tolerability issues QT interval prolongationLevofloxacin Broad, Increase in dose limited due to bactericidaltolerability issues Ciprofloxacin Gram negative, Increase in doselimited due to bactericidal tolerability issues Daptomycin Grampositive, Increase in dose limited due to toxicity bactericidalColistin, Gram negative, Only used as last resort antibiotics duePolymixin B bactericidal to renal and neurotoxicity G-, bactericidalTigecycline Broad, Increase in dose limited due to GI statictolerability issues Minocycline Broad, Increase in dose limited due toGI static tolerability issues

Also, a drug in vivo is metabolized and/or quickly diffuses out of thebloodstream, neither of which is applicable ex vivo. Accordingly, drugsmay be administered at lower doses extracorporeally since the entiredrug dose will remain in the blood or blood fraction.

A simplified example of the extracorporeal pharmaceutical module's “highin vitro concentration/low in vivo concentration” effect is as follows:A dose of an antimicrobial drug is 1 gram metered into a 1 literpharmaceutical module, which produces a high drug concentration of 1 mgper ml. If the volume of distribution for this drug is 100 liters, thenthe concentration in the human body would only be 0.01 mg/cc withouteven accounting for in vivo metabolic deactivation of the drug and/orits clearance from the body via the hepatic and renal systems. Ineffect, the total dose of the antimicrobial washing back into thepatient after transit through the high concentration pharmaceuticalmodule may be so minimal, that in many situations the patient will needto be administered some additional parenteral antimicrobial drug simplyto attain the appropriate minimum inhibitory concentration.

Appropriate initial ex vivo doses can be calculated by one of ordinaryskill in the art of pharmacokinetics based on the minimum inhibitory(MIC, IC₅₀), minimum effective (EC₅₀), and/or median lethal dose (LD₅₀)for any particular drug. Doses will also take into consideration theextracorporeal blood volume (e.g., about 0.5 to about 2 L), duration ofthe circuit, and age, gender, weight, and condition of the subject.

Exemplary pharmaceutical agents include, but are not limited to,antimicrobial, anti-viral, antibiotic, anti-fungal, and anti-parasiticdrugs. Specific exemplary pharmaceutical agents include, but are notlimited to, beta-lactams, sulfonamides, quinolones, aminoglycosides,carboxyquinolones (e.g., ciprofloxacin, levofloxacin, moxifloxacin),protein synthesis inhibitors, vancomycin and related drugs (e.g.,teicoplanin), ketolides, quinupristin and/or dalfopristin, linezolid,bacteriocins, mupirocin, anti-neoplastics, daptomycin, antiglycolytics,gluconeogenesis inhibitors, anti-metabolites (e.g., folate, pyrimidine,cytidine, purine), detergents (e.g., polymixin B, colistin),transitional metals, heavy metals, cycloserine, anti-fungals (e.g.,amphotericin B, fluocytosine, imidazoles, triazoles, echinocandins),fermentation inhibitors, anti-herpes virus agents (e.g., acyclovirfamily), anti-influenza agents (e.g., amantadine family, oseltamvir,zanamivir), anti-hepatitis agents (e.g., adefovir, interferon-alfa,lamivudine, pegylated interferon), ribavirin, imiquimod, cidofovir,anti-retroviral agents, protease inhibitors, anti-malarials (e.g.,quinine family, artemisinin family, atovaquone), eflornithine,melarsoprol, nitroimidazoles, pentamidine, sodium stibogluconate, otherancient antimicrobial agents to include heavy metal compounds, suramin,and benzimidazoles.

In some embodiments, more than one drug can be administered via a singlepharmaceutical module. In other embodiments, a single drug isadministered in each pharmaceutical module.

In some embodiments, multiple pharmaceutical modules are sequentiallyemployed to subject the pathogen to an orchestrated attack. For example,a first pharmaceutical module can employ agents that attack cell walls,membranes, or capsules. Gram positive bacteria such as MRSA possessthick cell wall capsules, which impede their uptake of drugs. Drugs suchas ionized silver can degrade microbial cell walls and/or cellmembranes. A pulsed electrical field module also may be used to increasebacterial pore size and allow antimicrobial agents to more easily enterthe pathogen's cytoplasm. Drugs such as daptomycin and vancomycin can beemployed to further attack the cell membranes and/or cell wall capsule.After this exposure, the a second pharmaceutical module can employ drugssuch as azithromycin or linezolid that will now more easily gain accessto the cytoplasm of the pathogen and attack critical microbial metabolicpathways for energy metabolism, protein synthesis, and reproduction.

In another embodiment, pharmaceutical modules are sequentially orsimultaneously paired with environmental control modules. For example,modules effecting aerobic conditions can be paired with pharmaceuticalagents effective in aerobic conditions. Additionally or alternatively,modules effecting anaerobic conditions can be paired with pharmaceuticalagents effective in anaerobic conditions. Exemplary pharmaceuticalagents effective in anaerobic conditions include, but are not limitedto, metronidazole and clindamycin. Exemplary pharmaceutical agentseffective in aerobic conditions include, but are not limited todaptomycin, beta-lactams, erythromycins, trimethoprim, andnitrofurantoin. Some drugs, such as ionic silver, can be effective ineither aerobic or anaerobic conditions (see, e.g., FIG. 14).

In another example of this embodiment in which a pharmaceutical moduleis paired with an environmental control module, the environmental modulecauses a decrease in the pH of the blood or blood fraction in order tomake the pharmaceutical agent more effective against the pathogen. Forexample, the pH may be decreased, and a quinolone, aminoglycoside, orbeta-lactam may be used. Both finafloxacin (8-cyano subclass) anddelafloxacin (which are in clinical development) show an increase inactivity at an acidic pH. Finafloxacin exhibits a 4- to 8-fold increasein activity (denoted by a 4- to 8-fold lowering of the MIC) at pH 6.0compared to activity at pH 7.4. A similar effect is observed withdelafloxacin. These changes in activity are likely to be a result, atleast in part, of a transmembrane diffusion model, in which uncharged(neutral or zwitterionic) species cross the membranes more easily thanthose having a net charge. At acidic pH, 50% of delafloxacin is under aneutral form, favoring its uptake over other agents.

In addition to small molecule pharmaceutical agents, other agents suchas bacteriocins can be used in the pharmaceutical module(s).Bacteriocins are small, heat stable proteins that are produced bynonpathogenic bacteria that attack disease causing bacteria. Theirspectrum of coverage is narrow, but the progress of rapid DNA-basedbacterial identification will allow precise utilization of these narrowspectrum antimicrobial compounds. For example, the bacteriocinlysostaphin can be employed in an extracorporeal pharmaceutical moduleto attack MRSA.

In one embodiment, the pharmaceutical agent is concentration dependent.This means that the higher its concentration, the more quickly andthoroughly it kills or impairs the pathogens. The system's ability tosafely produce and expose pathogens to super high concentrations of suchantibiotics will be maximized.

Suitable drugs include both bacteriocidal and bacteriostatic drugs. Insome embodiments, at least one bacteriocidal drug is administered. Inone embodiment, a bacteriostatic drug is administered, and another modeof attack, e.g., bacteriocidal agent or radiation, is also employed.

The extracorporeal system of the invention can optionally include amodule that removes all or some of the pharmaceutical agent before bloodreturn. This toxin removal module (see, e.g., FIG. 1) and describedfurther below can reduce the pharmaceutical agent concentration to atolerable in vivo dose. Alternatively, such a module can removesubstantially all of the pharmaceutical agent before blood return, whichmay be useful for agents that do not have a tolerable in vivo dose.

In one embodiment, the pharmaceutical agent is silver. Without beingbound by theory, the silver ion reacts with sulfhydryl or thiol groupsand has negative effects on microbial enzymes, proteins, cell walls, andcell membranes. Silver is a transitional metal as opposed to heavymetals such as lead, and therefore has less toxicity in humans, as itpreferentially destroys microbial cells. In one embodiment, thepharmaceutical module administers ionic silver, e.g., at concentrationsof about 0.01 to about 0.5 ppm, about 0.05 to about 4 ppm, about 0.1 ppmto about 0.3 ppm, or about 0.2 ppm. In this embodiment, the silver canbe introduced into the blood as silver chloride solution or via a simpleelectrolytic donor plate device.

3. Radiation Module

In some embodiments, the extracorporeal system of the invention mayinclude one or more radiation modules (FIG. 5). The radiation module candeliver X-ray, ultraviolet (UV), light emitting diode (LED), infrared(IR)(with and without riboflavin other potentiators), and visible,laser, and/or radiofrequency radiation. In one embodiment, the radiationmodule delivers X-ray and/or UV radiation. Like the pharmaceuticalmodules, multiple types or strengths of radiation may be delivered via asingle radiation module (See, e.g., FIG. 5), or multiple radiationmodules may be incorporated into the extracorporeal system.

Without being bound by theory, it is believed that radiation degradescell walls, thus potentially rendering the pathogens more susceptible topharmaceutical attack. Radiation also inhibits lymphocyte cytokinerelease, which will decrease cytokine-mediated shock in subjects.

In one embodiment, the blood (or blood fraction(s)) are subjected toabout 1 to about 500, about 5 to about 150, about 25 to about 100, orabout 50 to about 75 gray units of X-ray radiation.

As noted above, UV radiation inherently generates variable amounts ofheat, depending on the particular parameters used. Therefore, the UVradiation module in some embodiments, is sequentially or simultaneouslycoupled with an environmental module that decreases the temperature ofthe blood or blood fraction. In certain embodiments, the temperaturemodule is used to lower the temperature of the blood or blood fractionabout 5° C. to about 25° C., about 5° C. to about 15° C., about 10° C.to about 25° C., about 5° C. to about 10° C., about 8° C. to about 12°C., or about 8° C. to about 10° C.

4. Hydrostatic Compression Module

In certain embodiments, the extracorporeal system of the invention mayinclude one or more hydrostatic compression (high hydrostatic pressure(HHP)) modules (FIG. 6). An HHP module will consist of a special circuitor vessel that can be hermetically sealed and then pressurized via afluid pump capable of producing elevated extracorporeal pressure toachieve microbial inactivation. In certain embodiments, the pressureranges from about 50 MPa to about 1,100 MPa, about 50 MPa to about 1,000MPa, about 100 MPa to about 1,000 MPa, about 100 MPa to about 400 MPa,or about 400 MPa to about 1,000 MPa. The pressure may be applied as asingle continuous treatment, or as a series of sequential cycles.

In certain embodiments, HHP modules are sequentially or simultaneouslypaired with environmental control modules and/or other treatmentmodules. For example, the temperature may be modulated higher or lowerprior to, during, and immediately after HPP treatments, in order tomaximize antimicrobial effects and to avoid overheating of theextracorporeal fluids. In addition, pH modulation may also be utilizedwith the HHP module as titration of pH can positively impact HHPmicrobial inactivation.

A pharmaceutical module also may be included prior to, during, or afterpressurization with HHP, as a pharmaceutical agent can have synergisticantimicrobial effects when combined with increased fluid pressures. Inaddition, in certain embodiments, carbon dioxide is added to the fluidsin the HHP module as the presence of carbon dioxide dramaticallyenhances the antimicrobial effects of HHP treatments when added to thefluid being treated at or near its supercritical fluid phase (which canbe created in the module via modulation of temperature and pressure).Under these conditions, carbon dioxide can penetrate both bacterial cellwalls, as well as thick spore capsules, where it can abruptly decreaseintracellular pH and then, upon depressurization, extract the contentsof the microbial cell. In some embodiments, the pCO₂ levels used in theHHP module range from about 45 to about 200 mm Hg, about 45 to about 175mm Hg, or about 75 to about 150 mm Hg. In certain embodiments, the pCO₂levels used in the HHP module range from about 45 to about 150 mm Hg.

As noted above, HHP inherently generates variable amounts of heat,depending on the particular parameters used. Therefore, the HHP modulein some embodiments, is sequentially or simultaneously coupled with anenvironmental module that decreases the temperature of the blood orblood fraction. In certain embodiments, the temperature module is usedto lower the temperature of the blood or blood fraction about 5° C. toabout 25° C., about 5° C. to about 15° C., about 10° C. to about 25° C.,about 5° C. to about 10° C., about 8° C. to about 12° C., or about 8° C.to about 10° C.

HHP has variable effects at various pressurization levels.Pressurization levels below 400 MPa can cause changes to cellularmorphology and function that are reversible with depressurization. Oncecellular pressures exceed 400 MPa, the effects are irreversible, andinclude separation of the cell wall from cell membrane, loss of osmoticresponsiveness, enzymatically driven DNA shearing, and loss of protein,RNA, and metal ions to the extracellular fluid.

5. Pulsed Electric Field Module

In certain embodiments, the extracorporeal system of the invention mayinclude one or more pulsed electric field (PEF) modules (FIG. 7). PEFtreatment utilizes the application of high voltage pulses to manipulatethe cell membrane of a pathogen. In certain embodiments, the highvoltage pulse ranges from about 10 to about 100 kV/cm, about 10 to about80 kV/cm, about 20 to about 100 kV/cm, or about 20 to about 80 kV/cm. Incertain embodiments, the electrical pulse is applied for about 0.5 toabout 600 microseconds, about 0.5 to about 500 microseconds, about 1 toabout 600 microseconds, or about 1 to about 500 microseconds. Theelectrical pulse may be applied to a static or circulating fluid flowingin parallel, co-axial, or co-linear configurations within the module. Incertain embodiments, the fluid can be treated by the PEF module in astepwise circulation mode or a recirculation mode.

The PEF module is particularly suitable to the treatment of yeast andgram negative bacterial pathogens, although it is useful under for thetreatment of other pathogens as well. PEF mainly targets microbial cellmembranes, and its effects are reversible at lower levels of exposure.PEF causes disruption to the morphology and functional integrity of thecell membranes and their pores. At lower levels of energy, changes inthe microbial cell membranes are transient and reversible, whereashigher levels of energy will cause cell rupture or electroporation.Because the effectiveness of PEF depends upon pH, presence ofantimicrobial and ionic compounds, solution conductivity, ionicstrength, and growth phase of the microbes, in certain embodiments, thePEF module is sequentially or simultaneously coupled to one or moreenvironmental or other treatment modules (e.g., pharmaceutical module)to be most effective against pathogens. In one embodiment, lower levelsof PEF are applied and have the potential to facilitate delivery of manyantimicrobial agents, including ancient heavy metals into the cytoplasmof microbes, while defeating microbial resistance mechanisms such asefflux pumping. Although spores are relatively resistant to PEF,multiple sequential and/or simultaneous physical and chemical attackswithin the extracorporeal circuit should prove effective in degradinghardy spores.

As noted above, PEF inherently generates variable amounts of heat,depending on the particular parameters used. Therefore, temperaturemodulation may be necessary for PEF, both to adjust the growth phase ofmicrobes prior to initiation of treatments, and to cool theextracorporeal fluids during and after treatments. Accordingly, the PEFmodule in some embodiments, is sequentially or simultaneously coupledwith an environmental module that decreases the temperature of the bloodor blood fraction. In certain embodiments, the temperature module isused to lower the temperature of the blood or blood fraction about 5° C.to about 25° C., about 5° C. to about 15° C., about 10° C. to about 25°C., about 5° C. to about 10° C., about 8° C. to about 12° C., or about8° C. to about 10° C.

6. Microwave Module

In certain embodiments, the extracorporeal system of the invention mayinclude one or more microwave modules (FIG. 8). This module isparticularly suitable for the treatment of malarial pathogens in theblood or blood fraction. Plasmodium species parasitize red blood cells,and the parasitized cells adhere to vessel walls, causing stasis andinflammation (e.g., causing cerebral malaria when it occurs in the brainvasculature). Malarial parasites within red blood cells concentrate anddetoxify iron from hemoglobin molecules in a form (Fe+++) that rendersthe iron deposits susceptible to heating by microwave. The heat of themicrowave destroys both the parasite and the red blood cell. In certainembodiments, the microwave module is used at about 0.5 to about 25 cm,about 0.5 to about 20 cm, about 1 to about 25, or about 1 to about 20 cmwavelengths. In certain embodiments, the microwave module uses an energylevel of about 0.5 to about 20 W. In certain embodiments, the energylevel is about 1 to about 100 mW, or about 100 to about 200 mW. In someembodiments, the microwave module treats the fluid for about 50microseconds to about 15 seconds, about 50 microseconds to about 10seconds, about 100 microseconds to about 15 seconds, or about 100microseconds to about 10 seconds.

The selectively destroyed red blood cells (i.e., parasitized cells)and/or their contents (e.g., excess liberated iron) can then be removedby various treatments, including those discussed above or below, such asfiltration, centrifugation, chelation, adhesion, or microfluidicseparation techniques. Additional measures to remove free hemoglobinfrom the blood or blood fraction may be necessary, depending on thelevel.

Microwave inherently generates variable amounts of heat, depending onthe particular parameters used. Therefore, temperature modulation may benecessary for the microwave module to cool the extracorporeal fluidsduring and after treatments. Accordingly, the microwave module in someembodiments, is sequentially or simultaneously coupled with anenvironmental module that decreases the temperature of the blood orblood fraction. In certain embodiments, the temperature module is usedto lower the temperature of the blood or blood fraction about 5° C. toabout 25° C., about 5° C. to about 15° C., about 10° C. to about 25° C.,about 5° C. to about 10° C., about 8° C. to about 12° C., or about 8° C.to about 10° C.

7. Sonification Module

In certain embodiments, the extracorporeal system of the invention mayinclude one or more sonification modules (FIG. 9). Ultrasound is theenergy generated by sound waves of frequencies above the human hearingand is roughly defined by a frequency range from about 10 kHz to about1.5 GHz, about 10 kHz to about 1 GHz, about 18 kHz to about 1.2 GHz, orpreferably about 18 kHz up to about 1 GHz. Ultrasonic waves are createdby magnetostrictive or piezoelectric transducers, which transformelectrical energy into mechanical oscillations, and are transferred intothe treatment medium either directly via sonotrodes or indirectly incase of ultrasonic baths. The longitudinal sound waves can betransmitted into fluids causing cyclic compressions and rarefactions ofthe material. High-intensity, low-frequency (about 10 to about 150 kHz,about 16 to about 150 kHz, about 10 to about 100 kHz, or about 16 toabout 100 kHz) ultrasound can lead to cavitation, the creation, growth,and violent collapse of gas bubbles. The bubble collapse is accompaniedby high pressure and temperature peaks (up to about 100 MPa and about5000 K) as well as intense local shear. Such high power ultrasoundtreatments have the potential to improve mass transfer processes andheat transfer. In contrast, low intensities and high frequencies in theMHz-range lead to sonication treatments with acoustic streaming as themain mechanism. Such low energy ultrasound is used for non-destructivetesting as well as for the stimulation of living cells.

Cavitation and associated phenomena can reduce the use of chemicalsneeded for antimicrobial treatment. With respect to microorganisminactivation, the singular use of ultrasound is generally ratedinsufficient, but ultrasound is promising in combination with othertreatments because sonication induced cell damage leads to a highersensitivity towards other treatments. Therefore, in the disclosedsystems and methods, when a sonification module is included, typicallyone or more additional treatment modules are included to maximize theantimicrobial effect.

As noted above, sonification inherently generates variable amounts ofheat, depending on the particular parameters used. Therefore, thesonification module in some embodiments, is sequentially orsimultaneously coupled with an environmental module that decreases thetemperature of the blood or blood fraction. In certain embodiments, thetemperature module is used to lower the temperature of the blood orblood fraction about 5° C. to about 25° C., about 5° C. to about 15° C.,about 10° C. to about 25° C., about 5° C. to about 10° C., about 8° C.to about 12° C., or about 8° C. to about 10° C.

F. Toxin Removal Module

The extracorporeal system of the invention may include one or more toxinremoval modules, such as a filtration module, a dialysis module, achelation module, and/or an absorption or adsorption module, to removeboth pathogenic endotoxins as well as potentially toxic drugs, drugcomponents, or drug byproducts (FIGS. 10-13). In other embodiments, theextracorporeal system of the invention does not include a toxin removalmodule.

1. Filtration Module

In certain embodiments, the extracorporeal system includes one or morefiltration modules. A filtration module may include conventional meansfor capturing pathogens and/or other toxins while permitting other bloodcomponents to pass through. The filter can be a mechanical filter suchas a screen or membrane or it can include an affinity-based method ofcapture such as with antibody capture resins (e.g., monoclonalantibodies with magnetic nanoparticles). In certain embodiments, thefilter is a polymyxin cartridge which removes bacterial endotoxins fromthe blood or blood fraction. Filtration and removal of intact bacterianot only decreases bacterial load, but also reduces or eliminates theproduction of endotoxins, which are potentially lethal.

Nevertheless, especially for modules including pharmaceutical and/orradiation modules, in one embodiment, the filtration module may includean endotoxin filter, such as a polymyxin cartridge (See FIG. 10).

In another embodiment, the filter can be a ceramic filter having anaverage pore size of about 0.1 to about 5 microns, about 0.3 to about1.5 microns, or about 1 micron. In certain embodiments, the ceramicfilter has an average pore size of about 0.05 to about 0.15 microns, andthe filter can trap a virus. In certain other embodiments, the ceramicfilter has an average pore size from about 1.5 to about 5 microns, andthe filter can trap bacteria. In certain other embodiments, the ceramicfilter has an average pore size from about 12 to about 50 microns, orlarger, and the filter can trap parasites and fungi.

In another embodiment, the filtration module can utilize an electricalcharge gradient to move the blood across the filter (i.e.,electrodialysis). Gram positive bacteria have negative surface chargesdue to their carboxyl and phosphate capsular components. This allowsthem to migrate through the membrane towards a positive electrodialyticcharge, and then be sequestered and eliminated from the circuit.

In one embodiment, whole blood, or one or more blood fractions, arepassed through one or more filtration modules. In one embodiment, aplasma fraction is passed through a filtration module utilizing aceramic filter. Additionally or alternatively, a red blood cell fractioncan be diluted with normal saline then, as an alternative tocentrifugation, be subjected to bacterial filtration by electrodialysis,through a membrane having an average pore size of about 0.1 to about 2microns, about 0.2 to about 2 microns, about 0.3 to about 2 microns,about 0.1 to about 1.5 microns, about 0.2 to about 1.5 microns, or about0.3 to about 1.5 microns.

2. Dialysis Module

In certain embodiments, the extracorporeal system includes one or moredialysis modules (FIG. 11). In certain embodiments of the presentextracorporeal system, antimicrobial molecules (e.g., antibiotics) willbe used in the treatment module that would have deleterious effects on asubject if the molecules were introduced into the subject. Therefore,prior to returning the blood or blood fraction to the subject, the bloodor blood fraction is subjected to dialysis in order to reduce theconcentration of the antimicrobial molecules. Whether a particularantimicrobial molecule may be removed by dialysis is dependent at leastin part on molecular weight, charge, and albumin binding. In certainembodiments, the dialysis is conducted for about 2 to about 6 hours,about 3 to about 5, or about 4 to about 5 hours. In certain embodiments,the antimicrobial molecule concentration is reduced to about 5% to about50% of its pre-dialysis concentration, and the remaining molecules maybe cleared by the subject's kidneys. Examples of antimicrobial moleculesthat may effectively be dialyzed from the blood or blood fraction areshown in Table 3 (extracted fromhttp://www.clinicaldruguse.com/dialysisDrugs.php accessed on Dec. 28,2011).

TABLE 3 Percent removed Percent removed Drugs By hemodialysis By CAPDAntimicrobial Agents

 Aminoglycosides Aminoglycosides 50% 20-50% Spectinomycin 50% —

 Carbapenems Biapenem 90% — Imipenem 80-90% Negligible Meropenem 50-70%—

 Cephalosporins Cefaclor 33% — Cefadroxil 50% — Cefamandole 50%Negligible (5%) Cefazolin 50% 20% Cefipime 40-70% 26% Cefmenoxime 16-51%Negligible (<10%) Cefmetazole 60% — Cefodizime 50% Negligible (15%)Ceforanide 20-50% Negligible Cefotaxime 60% Negligible (5%) Defotiam30-40% — Cefoxitin 50% Negligible Cefpirome 32-48% Negligible (12%)Cefpodoxime 50% — Cefprozil 55% — Cefroxadine 50% — Cefsulodin 60% —Ceftazidime 50% Negligible Ceftibuten 39% — Ceftizoxime 50% Negligible(16%) Ceftriaxone 40% Negligible (4.5%) Cefuroxime — 20% Cephacetrile50% — Cephalexin 50-75% 30% Cephalothin 50% — Cephapirin 20% —

 Monobactams Aztreoman 40% Negligible Carumonam 51% — Moxalactam 30-50%Negligible (15-20%)

 Nitroimidazoles Metronidazole 45% Negligible (10%) Ornidazole 42%Negligible (6%) Tinidazole 40% —

 Oxaxolindiones Linezolid 33% —

 Penicillins Amdinocillin 32-70% Negligible (<4%) Amoxicillin 30% —Ampicillin 40% — Azlocillin 30-45% — Carbenicillin 50% — Mezlocillin20-25% 24% Penicillin 50% — Piperacillin 30-50% — Temocillin 50%Negligible (6%) Ticarcillin 50% Negligible

 Sulfonamides Sulfamethoxazole 50% Negligible (8%) Trimethoprim 50%Negligible (7%)

 Antifungals Fluconazole 40% Negligible (18%) Flucytosine 50% —

 Antituberculous Agents Para-aminosalicylic Acid 50% — Isoniazid 75% —

 Antiviral Agents Abacavir 24% — Acyclovir 60% Negligible (<10%)Cidofovir 50% Negligible Didanosine 20-67% Negligible Vidarabine 50% —

3. Chelation Module

In certain embodiments, the extracorporeal system includes one or morechelation modules (FIG. 12). In certain embodiments of the presentextracorporeal system, heavy metal legacy antimicrobial compounds willbe used in the treatment module that would have deleterious effects on asubject if the compounds were introduced into the subject. Legacyantimicrobial substances include heavy metal and transitional metalagents such as arsenic, mercury, antimony and lead that were used invarious forms. These compounds possessed very significant antimicrobialeffectiveness, but were limited by their systemic toxicity in humans.Therefore, prior to returning the blood or blood fraction to thesubject, the blood or blood fraction is contacted with a chelating agentin order to inactivate the antimicrobial molecules.

Chelating agents are chemical compounds that are capable of tightlybinding the ions of such metallic antimicrobial preparations andinactivating their toxic effects. The inactivated chelated metal ioncomplexes are water soluble and are able to be eliminated from the bodyvia the patient's kidneys. The first practical chelating agent for heavymetal poisoning was developed during world War I to combat poisoningproduced by weaponized arsenical gas preparations, and was known as BAL,or British Anti-Lewisite. The disclosed extracorporeal systems willallow for further practical research and development of new forms ofmetallic antimicrobial agents, in order to provide increasedeffectiveness against resistant microbial pathogens.

Suitable chelation agents are any that are known to one of ordinaryskill in the art, and many are currently commercially available for usein the treatment of poison victims, such as BAL (for use with, e.g.,lead, mercury, arsenic), dimercaptosuccinic acid (DMSA)(for use with,e.g., lead, mercury, cadmium, arsenic), desferal,2,3-dimercapto-1-propanesulfonic acid (DMPS)(for use with, e.g., severeacute arsenic and mercury poisoning), pencillamine (for use with, e.g.,copper, gold, lead), ethylenediaminetetraacetic acid (EDTA)(for usewith, e.g., lead), deferoxamine (for use with, e.g., iron), deferasirox(for use with, e.g., iron), thiobendazole (for use with, e.g.,antimony), and alpha lipoic acid (ALA).

4. Absorption Module

In certain embodiments, the extracorporeal system includes one or moreabsorption modules (FIG. 13). Adsorbent filters, cartridges, or columnsmay be used to physically bond to and retain toxins on their surface.Absorbent filters, cartridges, or columns may be used to chemicallyintegrate the toxin molecule into the structure of the filter,cartridge, or column. Any adsorbent or absorbent filter material knownto one of skill in the art may be used. In certain embodiments, theadsorbent or absorbent filter, cartridge, or column comprises a materialthat is selected from a group that includes, but is not limited toactivated charcoal, soda lime, and polymixin. In certain embodiments,the adsorption or absorption filter, cartridge, or column is used toremove the toxin or other unwanted non-dialyzable substances from thefluid, and the filter, cartridge, or column is discarded. In oneembodiment, the absorbent material may be soda lime, and it may be usedto remove carbon dioxide from the fluid. In another embodiment, theadsorbent material is polymixin, and it may be used to adsorb bacterialendotoxins to the polymixin coated fibers, which are then discarded.

G. Blood Return

Blood return, like blood removal described above, can be accomplished byany conventional means, e.g., catheter or port, known in the art. Theblood return port and the blood removal port can enter the patient atthe same approximate location (e.g., the same arm as depicted in FIG.1), or the blood removal port and blood return may have entirelyindependent entry points.

II. Research Tools

The extracorporeal system and methods of the invention may be used forresearch purposes in which case, the subject may be a small mammallaboratory animal such as a mouse, rat, or rabbit. In other embodiments,the subject may be replaced by a non-living blood source, such as ablood reservoir (containing e.g., fresh animal blood), to create afree-standing, closed circuit.

The extracorporeal systems and methods of the invention may be used todevelop new drugs or new classes of drugs, screen drugs to assess theextracorporeal efficacy under various conditions. In particular, theinvention may be used to assess and optimize treatment with drugs thatare effective in conditions of low glucose, anaerobic, or otherfermentative conditions.

The invention can also be used for culturing pathogens. Culturingpathogens in this device allows for continuous control of variousenvironmental factors as well as the added advantage of mimickedcirculation and other in vivo effects, such as the creation of, andtreatment for, biofilms.

Without further elaboration, it is believed that one skilled in the artusing the preceding description can utilize the invention to the fullestextent.

The embodiments described above are merely illustrative and are notmeant to be an exhaustive list of all possible embodiments,applications, or modifications of the invention. Thus, variousmodifications and variations of the described methods and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in biology or in the relevant fields are intended to bewithin the scope of the appended claims.

The disclosures of all references and publications cited above areexpressly incorporated by reference in their entireties to the sameextent as if each were incorporated by reference individually.

What is claimed is:
 1. An extracorporeal method for treating ablood-borne disease comprising the steps of: a) removing blood from asubject; b) contacting the blood with an anticoagulant; c) optionallyseparating the blood into two or more blood fractions; d) optionallymodifying the environment of the blood or a blood fraction; e) treatingthe blood or blood fraction with cold plasma; f) treating the blood orblood fraction with hydrostatic pressure, a pulsed electrical field, apharmaceutical agent, centrifugation, microwave, radiation,sonification, or a combination thereof; and g) returning at least aportion of the blood or blood fraction to the subject.
 2. Anextracorporeal system comprising: a) a blood removal port; b) ananticoagulant module; c) a module adapted to modify the environment ofblood or a blood fraction; d) a treatment module adapted to administercold plasma; e) one or more treatment modules adapted to administerhydrostatic pressure, a pulsed electrical field, a pharmaceutical agent,microwave, centrifugation, radiation, sonification, or a combinationthereof; and f) a blood return port.